Hepatocellular carcinoma is a human cancer with a poor prognosis. Indeed, most patients diagnosed with hepatocellular carcinoma do not live for even a year following diagnosis.
Cytotoxic agents such as phosphoramidc mustard (PM) are highly toxic to the malignant cells of the liver, i.e. the hepatoma cells. However, due to its high polarity and chemical instability, PM possesses a short circulation half life and rapid urinary excretion in vivo. Consequently, PM itself is not clinically useful as an anticancer agent for hepatocellular carcinoma. Attempts have been made to overcome the polarity problems of PM by encapsulating this cytotoxic agent into liposomes. However, such liposomes tend to accumulate in the reticuloendothelial system in liver and spleen, thereby limiting their usefulness as vehicles for delivering cytotoxic agents to hepatoma cells.
Hepatoma cells are known to express high levels of insulin receptor as compared to normal hepatocytes and other normal cells. Thus, the insulin receptor is an attractive target for delivering cytotoxic agents to hepatoma cells. Previously, it has been demonstrated that liposomes that have NB1-palmitoyl insulin incorporated therein are selectively taken up by hepatocytes in rodents. Unfortunately, the procedure used for making such liposomes is tedious and time-consuming. Moreover, the procedures currently used to prepare the lipid-modified insulin requires lengthy protection and purification steps. This method, which involves selective modification of reactive sites on the insulin molecule, usually is achieved by blocking other undesired sites. Thus, when synthesizing NB1 palmitoyl insulin or NB29 palmitoyl insulin, undesired free amino groups, typically, are first blocked with t-boc or other similar groups, followed by purification of the insulin derivatives with only the desired reactive amino group open. The purified species is then reacted with palmitic acid hydroxysuccinamide ester. The preparation is completed by deblocking the t-boc groups to free the other unmodified amino groups followed by final purification. Because the synthesis involves a multiple-step reaction, separation, dialysis and lyophylization, the yield of this method is extremely low.
Accordingly, it is desirable to have a modified insulin that can bind to the insulin receptor and that can be incorporated into liposomes for targeting cytotoxic agents to hepatocellular carcinoma cells. A simple method for making the modified insulin is also desirable.